Bordetella trematum bacteraemia secondary to an empyema in an immunocompromised host: A case report and review of the literature

Introduction. Bordetella trematum infection remains uncommon. More cases of bacteraemia are reported in recent years with the primary infection largely originating from skin and soft tissue sites. Yet, our understanding of its virulence, antibiotic susceptibility profile and treatment is still limited. Case presentation. Case presentation. We report the first case of B. trematum bacteraemia from a left-sided empyema. An 87-year-old female patient with a past medical history of ischaemic heart disease, diabetes mellitus complicated by nephropathy and locally advanced left breast adenocarcinoma presented with fever, productive cough and shortness of breath. The B. trematum isolates from blood and pleural fluid were identified by MALDI-TOF and 16S rRNA sequencing. Ceftriaxone and azithromycin commenced empirically on admission were switched to piperacillin-tazobactam after 2 days due to lack of clinical improvement. Despite a pleurocentesis and 1 week of piperacillin-tazobactam with microbiological clearance in blood, the patient continued to deteriorate. Decision to withdraw treatment was made in view of the patient’s prognosis, and the patient succumbed on the fourteenth day of admission. The isolate was susceptible to piperacillin-tazobactam, imipenem and meropenem but had reduced susceptibility or was non-susceptible to cefuroxime, cefotaxime, ceftazidime, cefepime, the aminoglycosides and fluoroquinolones. Conclusion. Invasive B. trematum infection is associated with significant mortality. Consensus for antibiotic treatment remains unclear, with limited susceptibility data to support specific antibiotic use. We expect more clinical cases will surface with improved microbial identification systems, as well as enhanced clinical awareness. Standardized and more robust susceptibility work are needed to provide clear recommendations and establish consensus in treating invasive infections.


BACKGROUND
Bordetella trematum is an aerobic non-lactose fermenting Gram-negative bacillus, first recognized and described in 1996 by Vandamme et al. [1], Most published B. trematum cases [2][3][4][5][6] between 2004 to 2019 were based on the recovery of isolates from infected lower limb wounds in the presence of other pathogens, with only a handful of cases recovered from sterile sites. In 2020, Kukla et al. [7] described the recovery of B. trematum from the respiratory tract of an immunocompromised host, and more recently a letter-to-the-editor by Lee et al. [8] described a case of B. trematum catheter related bloodstream infection. To date, documented clinical cases are relatively scarce, limited to case series and reports. There are still gaps in our understanding of its pathogenesis and its potential virulence in causing infection.
We present a case of B. trematum bloodstream infection secondary to an empyema in an immunocompromised host, and the microorganism's antibiotic susceptibility profile. To our knowledge, this is the first published case of B. trematum bacteraemia from an empyema.

CASE PRESENTATION
An 87-year-old female with a past medical history of ischaemic heart disease, diabetes mellitus complicated by nephropathy, and locally advanced left breast adenocarcinoma presented with fever, productive cough and shortness of breath of 5 days' duration. The patient had also complained of reduced urine output during the same period.
At presentation, the patient was afebrile and alert, but tachypnoeic with a respiratory rate of 32 breaths per minute and oxygen saturation was 90 % on room air. Respiratory examination revealed reduced air entry on the left base. The patient's breast tumour was visible on the left chest, with a discharging blood-stained wound. The liver function test was unremarkable. Admission chest x-ray showed a moderate sized left pleural effusion. In the context of the COVID-19 pandemic and upper respiratory tract symptoms, a nose and throat swab was sent for respiratory pathogens detection. The multiplex PCR (Biofire Respiratory Panel 2.1 Plus) did not detect any viruses including SARS-CoV-2, nor any atypical pathogens (Bordetella pertussis, Bordetella parapertusis, Chlamydia pneumoniae and Mycoplasma pneumoniae).
The patient was empirically commenced on intravenous ceftriaxone 2 g once daily and oral azithromycin 500 mg once daily for treatment of complicated pneumonia with type II respiratory failure. With little clinical improvement, on the third day the empiric antibiotics were escalated to intravenous piperacillin-tazobactam 4.5 g twice daily (adjusted according to the patient's renal impairment).
The aerobic blood culture collected on admission (day 1) flagged positive at 24 hours of incubation in the automated blood culture system, BD BacTec FX System, and Gram stain revealed a Gram-negative bacillus. The following day, culture plates (blood and MacConkey agar) grew oxidase negative, non-lactose fermenting Gram-negative bacilli. Identification was performed by MALDI-TOF Vitek-MS and yielded the organism, B. trematum.
On the third day, a left pleurocentesis was performed for diagnostic and symptomatic relief. The appearance of the aspirated pleural fluid was yellow and turbid on macroscopic examination, with a pH of 7.46, cell count of greater than 10 000 cells/mm 3 (90 % neutrophil predominant), total protein of 26.3 g/l, glucose of 1.0 mmol/l, lactate dehydrogenase (LDH) of 1938 U/l and adenosine deaminase (ADA) of 35.9 U/l [normal range ≤ 30 U/l]. Gram-negative bacilli were seen on direct microscopy, and the pleural fluid cultured pure growth of B. trematum the following day, identified by MALDI-TOF. The biochemical, microbiological and clinical findings were in keeping with a left-sided empyema. The microbial identity of both isolates were confirmed by 16S rRNA gene sequencing.
Susceptibility testing was performed using the commercial broth microdilution panel, MicroScan Negative Panel (NM44) and supplemented with antibiotic gradient strip, E-test. The categorical susceptibilities reported were based on the non-Enterobacterales Clinical and Laboratory Standards Institute (CLSI) breakpoints. For the blood isolate, piperacillin-tazobactam was reported as susceptible with a MIC of 4 µg/ml, whilst the cephalosporins (cefotaxime, ceftazidime, cefepime) exhibited high MICs, between 8 to 32 µg/ml, falling within the susceptible and non-susceptible categories. The aminoglycosides (gentamicin, amikacin and tobramycin) and the fluoroquinolones (ciprofloxacin, levofloxacin and moxifloxacin) demonstrated reduced susceptibilities. Tetracycline (MIC <= 4 µg/ml) and trimethoprim-sulfamethoxazole (MIC <= 2 µg/ml) both tested susceptible. Co-amoxiclav and azithromycin were also tested given the clinical experience of these antibiotics used in treating other Bordetella species infection, both antibiotics recorded a MIC of 4 µg/ml. (Table 1).
Despite active treatment with piperacillin-tazobactam and microbiological clearance in two sets of follow-up blood cultures (collected on day 4 and day 5), the patient's clinical prognosis maintained guarded with progressively worsening renal function and type II respiratory failure. A collective medical and family decision was made to minimize further active intervention. Supportive and palliative care was instituted and the patient succumbed on the fourteenth day of admission. See Fig. 1 for a summary of events.

DISCUSSION
Since the discovery and recognition of B. trematum from clinical specimens, its pathogenic role in human infection has been debated over the years. Earlier case reports in the literature, were largely from skin and soft tissue infections of the ear, lower limb ulcers and wounds of diabetic patients, which were polymicrobial in nature [2][3][4][5][6]. Some may argue that B. trematum recovered from chronic lower limb ulcers or wounds were transient benign colonizers and may not have warranted immediate clinical attention [2]. Furthermore, reports of bloodstream infections [9,10] and peritonitis [11] were polymicrobial in nature where B. trematum was recovered as part of a mixed culture in the sample. In addition, Kulka et al. [7] suggested that the recovery of B. trematum in the presence of oropharyngeal flora from the respiratory tract (sputum and bronchoalveolar lavage) was a plausible source of infection in an oncology patient.
Two cases of bacteraemia reported by Majewski et al. [12] and Lacasse et al. [13] had originated from infected skin and soft tissue sites, but resulted in very different clinical outcomes. Majewski et al. described a fatal case from a rapidly progressive deep tissue  Since 2015, there has been a cumulative number of B. trematum bacteraemia cases reported (Table 2). Yet, relatively little is known about the pathogenesis, spectrum of disease and its antibiotic susceptibility profile. Studies have looked at its genomic sequences, described genes encoding the cytolethal distending toxin (CDT) and analysed the O-chain subunit of the lipopolysaccharide (LPS, endotoxin) [14][15][16]. Novikov et al. [17] reported a modification in lipid A of B. trematum, which may play a role in evading the host's immune system.
Host factors such as diabetes mellitus and underlying malignancy featured in most reported cases, reflecting a relatively susceptible host status as a pre-requisite for acquiring B. trematum infection. Wounds from long-standing ulcers or vascular exit sites have been hypothesized as likely entry sites into the bloodstream. Whilst colonization from the immediate environment has been proposed and transient host colonization is plausible, environmental niches have not been fully identified to truly establish the nature of this organism's portal entry into a human host [18].
In our case, the recovery of B. trematum was unlikely an environmental contaminant or colonizing flora, and we believe it had contributed to our patient's clinical outcome. Firstly, we recovered our isolates from two different sterile sites in pure culture, where each sample was collected independent from each other, 2 days apart. One was from the admission blood culture and the other from a day 3 pleural tap. Secondly, each sample was processed independent of each other in the laboratory, and at different time points. We believe the likely portal entry of B. trematum is the discharging left breast wound, given the contiguous anatomical site. However, no microbiological samples were obtained from this site nor any additional imaging performed to demonstrate direct invasion into the pleural space to support our suspicion. We recognize that B. trematum has true potential as an opportunistic invasive pathogen in an immunocompromised host, as evident in our case who was relatively immunocompromised secondary to advanced age, breast cancer, diabetes and chronic kidney disease.
MALDI-TOF and 16S RNA sequencing are methods for reliable identification of B. trematum in a clinical laboratory. Biochemical identification systems had led to misidentification in the past [2,3,5], which may have contributed to B. trematum isolates being overlooked in clinical specimens. With more clinical laboratories adopting the MALDI-TOF as their primary microbial identification system, B. trematum could be more readily and rapidly recognized. However, due to its rarity of occurrence from clinical sites and its relatively unknown pathogenicity, the reporting laboratory may dismiss it from mixed wound cultures, or may elect for confirmatory testing potentially delaying clinical reporting [19].
To date, there has not been clear consensus on specific antibiotic for treatment. Published susceptibility data for B. trematum are limited to case reports and studies to derive meaningful conclusion on treatment outcomes. In addition, the lack of speciesspecific breakpoints, different laboratory methodologies (i.e. disc diffusion, gradient strip diffusion, broth microdilution) and standards varied across published reports. Categorical susceptibilities reported in the literature were based on pharmacokineticpharmacodynamic (PK-PD) breakpoints from European Committee on Antimicrobial Susceptibility Testing (EUCAST), non-Enterobacterales CLSI breakpoints or Food and Drug Administration (FDA), to guide clinicians on therapeutic options. Whilst the mainstay of treatment for infected ulcers and wounds is surgical intervention, bloodstream and other sterile site infections will warrant systemic antibiotic treatment. Table 3 summarizes published bacteraemia cases and their phenotypic susceptibility profiles.
Buechler et al. [20] recognized the scarcity of published susceptibility data, and presented the molecular and phenotypic susceptibility profile of three unique clinical B. trematum isolates. Our B. trematum isolates demonstrated in vitro susceptibility with piperacillin-tazobactam, meropenem and imipenem, but exhibited reduced susceptibility to the cephalosporins, aminoglycosides and fluoroquinolones tested, echoing the phenotypic susceptibility findings by Buechler et al. The team had also identified the fluoroquinolone and aminoglycoside resistance genes in its isolates, and a beta-lactamase encoding gene. In light of this, we  Vitek-2, GSD (CLSI) DD, BMD (CLSI, FDA) GSD (EUCAST PK-PD) Continued had followed on with additional phenotypic testing to detect the presence of extended spectrum beta-lactamase (ESBL) by combined-disc testing (with cefotaxime and cefotaxime-clavulanate, and ceftazidime and ceftazidime-clavulanate), but did not detect the presence of ESBL in our isolate.
There may be limitations in the methodology used in susceptibility testing, as seen between our two B. trematum isolates. The one dilution difference in the cephalosporins and aminoglycosides, and the discordant results of the fluoroquinolones (levofloxacin and moxifloxacin) could be from intra-laboratory variations, or potentially, inherent challenges of susceptibility testing and interpretation with this organism.
Five of the eight bacteraemia cases reported had piperacillin-tazobactam as part of, or a definitive antibiotic treatment, of whom three survived. In our case, the B. trematum bloodstream infection was not sustained after a 7 day course of piperacillintazobactam, implies that other factors may have contributed to the patient's clinical deterioration. Current in vitro data supports the use of carbapenems and to some extent piperacillin-tazobactam as empiric agents when faced with a clinical infection, but clinical outcome data is necessary before recommendations can be issued. However, in the absence of available susceptibilities, we would probably not recommend cephalosporins, aminoglycosides or fluoroquinolones as first-line agents.

CONCLUSION
Invasive B. trematum infection remains uncommon but is associated with significant mortality in the immunocompromised. Consensus for antibiotic treatment remains unclear, with limited susceptibility data to support specific antibiotic use. We predict that this opportunistic pathogen will be reported more readily from clinical specimens with improved microbial identification systems, as well as enhanced clinical awareness. Standardized and more robust susceptibility works are needed to provide clear recommendations and establish consensus in treating invasive infections.

Funding information
There are no sources of funding received.
Author contributions C. Wong -writing -original draft, review and editing, data analysis. L.G. Calungsud -clinical data collection. M.V. La -data curation and analysis, nucleotide sequence submission.

Conflicts of interest
There are no potential conflict of interest.

Consent to publish
A written informed consent was obtained from the patient's next of kin for publication of this case report. A copy of the written consent is available for review by the Editor-in-Chief of this journal on request.  Thank you for submitting your paper to Access Microbiology. It has now been reviewed and I would like you to revise the paper in line with the reviewers' reports and any Editorial Office requirements below. The reviewer reports can be found at the bottom of the email.

Editor comments:
The work presented is clear and the arguments well formed.
This study would be a valuable contribution to the existing literature.
This is a study that would be of interest to the field and community.
Thank you for your submission. The manuscript is in a good state and while a recommendation of minor edits has been made, the edits are almost entirely typographically. Please address reviewers concerns.
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Please submit the revised version of your manuscript by 08/07/2023.
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Reviewers' comments and responses to custom questions:
Please rate the quality of the presentation and structure of the manuscript In this manuscript, the authors presented a case report of an 87-year-old female patient with bacteremia from a left-sided empyema. Bordetella trematum was isolated from blood and pleural fluid and despite the antibiotic treatment, the patient died 14 days after admission.

Description of the case(s)
Line 1: as this is the first time the name of the bacterium appears, I suggest not abbreviating the genus.
Line 1 -Changed to reflect "Bordetella trematum" Line 7: to designate the biological sex, it is better to use the term "female patient" instead of "lady".
Line 18 -Changed to reflect "female patient" Line 34: as the term "Bordetella trematum" appears in the title, a different keyword could be used instead.

Presentation of results
Line 57: there is a typo in "tachypnoeic" Line 58 -Written based on the British spelling "tachypnoeic". Changes made throughout the manuscript to reflect "Gram negative" Line 91: as the first sentence of this paragraph still refers to the bacterial identification test, it can be added to the previous paragraph. -Line 94 -Move this sentence to the previous paragraph.
Lines 107-108: what was the cause of death?
Line 114 -Pneumonia and empyema with multi-organ dysfunction, in the background of locally advanced breast cancer and poor co-morbidities. A decision to withdraw active treatment was made, and she received supportive care from day-11 of her hospital stay.
3. How the style and organization of the paper communicates and represents key findings A timeline of key case events can help visualize the course of the infection.
Line 216 -A visual time line to show the chronology of events added to the manuscript.

Literature analysis or discussion
Line 91: as the first sentence of this paragraph still refers to the bacterial identification test, it can be added to the previous paragraph.
(as above) Lines 107-108: what was the cause of death?
Lines 120-121: I suggest removing the phrase "it is therefore not unexpected for sceptics to question the role of B. trematum as a pathogen in its own right".
Line 127-128 -This sentence has been removed.
Lines 123-127: could the different clinical conditions of the patients have led to different outcomes? What are the similarities and differences with this case report?
Lines 130-134 -The two bacteraemia cases originated from skin/soft tissue sites, one with a rather dramatic clinical presentation/ deterioration followed by death vs the other case who attained complete clinical resolution.
There were many factors in Majewski's case that could have led to the demise of the patient.
However, during my review of published articles, (see summary of cases in Table 2 of the manuscript), what struck me was the poorer outcome in those with diabetes (3 out of 4 of the cases with diabetes died). These are merely personal observations, and one can't come to any conclusive association based on a few cases. More clinical data points are necessary.
I highlighted these two cases specifically because they had relatively matched underlying renal and cardiac conditions -with the exception of diabetes present in Majewski's, and CLL in Lacasse's case, which may or may not have contributed to the final clinical outcome.

Any other relevant comments
Line 241: Is there any ethical approval?
Line 284 -Written informed consent to publication was obtained from the patients' next of kin.
Ethical approval for single case reports is not required by the hospital, In several parts of the text terms such as "she/her" are used referring to the patient. I suggest replacing to "the patient".
-Changes made throughout the manuscript Table 2: in items 7 and 8, age/sex column is missing the letter y after age.  The authors report a case of an 87 year old female patient with a Bordetella trematum infection. The patient succumbed 14 days after admission and antibiotic sensitivity screening was carried out on the isolate and the author raise concerns about limited literature knowledge on standard treatment approaches.
The manuscript is generally well written, some minor comments below and the literature review and summary tables specifically will be of use to the community.

Minor comments
Line 1: Use full bacterial name in title. This will make accessing the paper easier and is good practice.
Line 62: please the Greek "μ" for micro litre. Here and throughout. iThenticate report